Reducing Enrichment Cultures Research Articles

Composition and Metabolic Potential of Fe(III)-Reducing Enrichment Cultures of Methanotrophic ANME-2a Archaea and Associated Bacteria

The key microbial group involved in anaerobic methane oxidation is anaerobic methanotrophic archaea (ANME). From a terrestrial mud volcano, we enriched a microbial community containing ANME-2a, using methane as an electron donor, Fe(III) oxide (ferrihydrite) as an electron acceptor, and anthraquinone-2,6-disulfonate as an electron shuttle. Ferrihydrite reduction led to the formation of a black, highly magnetic precipitate. A significant relative abundance of ANME-2a in batch cultures was observed over five subsequent transfers. Phylogenetic analysis revealed that, in addition to ANME-2a, two bacterial taxa belonging to uncultured Desulfobulbaceae and Anaerolineaceae were constantly present in all enrichments. Metagenome-assembled genomes (MAGs) of ANME-2a contained a complete set of genes for methanogenesis and numerous genes of multiheme c-type cytochromes (MHC), indicating the capability of methanotrophs to transfer electrons to metal oxides or to a bacterial partner. One of the ANME MAGs encoded respiratory arsenate reductase (Arr), suggesting the potential for a direct coupling of methane oxidation with As(V) reduction in the single microorganism. The same MAG also encoded uptake [NiFe] hydrogenase, which is uncommon for ANME-2. The MAG of uncultured Desulfobulbaceae contained genes of dissimilatory sulfate reduction, a Wood-Ljungdahl pathway for autotrophic CO2 fixation, hydrogenases, and 43 MHC. We hypothesize that uncultured Desulfobulbaceae is a bacterial partner of ANME-2a, which mediates extracellular electron transfer to Fe(III) oxide.

Characterization of Fe(III)-Reducing Enrichment Cultures and Isolation of Enterobacter sp. Nan-1 from the Deep-Sea Sediment, South China Sea

To characterize the Fe(III)-reducing bacteria, enrichment cultures were initiated by inoculating deep-sea sediment from the South China Sea (SCS) into the media with hydrous ferric oxide (HFO) as the sole electron acceptor. As indicated by Meta 16S rDNA Amplicon Sequencing, the microorganisms related to Fe(III)-reduction in the enrichment cultures were mainly Shewanella and Enterobacter. A new facultative Fe(III)-reducing bacterium was obtained and identified as Enterobacter sp. Nan-1 based on its 16S rRNA gene sequence and physiological characterizations. Enterobacter sp. Nan-1 was not only a mesophilic bacterium capable of reducing HFO with a wide range of salinity (4, 34, 40, 50 and 60 g L−1) efficiently, but also a piezotolerant bacterium that can proceed Fe(III)-reduction sustainedly at hydrostatic pressures between 0.1 and 50 MPa using glucose and pyruvate as carbon source. Furthermore, the geochemical characteristics of deep-sea sediment indicated that the microbial metabolism and iron reduction both remain active in the well-developed Fe(III)-reducing zone where the strain Nan-1 was obtained. To our knowledge, Enterobacter sp. Nan-1 could serve as a new applicative Fe(III)-reducing bacterium for future investigation on the iron biogeochemical cycle and diagenetic process of organic matter in the deep-sea environment.

The Influence of O2 on the Enrichment and Activities of Acidophilic Fe(III) Reducing Bacteria

Microbiological Fe(III) reduction under oxic and acidic conditions in acid mine drainage (AMD)-impacted systems is surprising, given the greater energetic favorability of O2 reduction than Fe(III) reduction. A possible explanation for this observation is that the energetic favorability of Fe(III) reduction approaches that of O2 reduction at pH < ∼2. However, some microorganisms require O2 for optimal Fe(III) reduction, and the process has still been observed at pH ≥ 3.5 under oxic conditions. We set up acidophilic Fe(III) reducing enrichment cultures with and without O2. The time of initiation and extent of Fe(III) reduction in oxic and anoxic enrichments were identical, and Fe(III) reduction by the enrichment cultures occurred to similar extents with subsequent transfers of the cultures. Desulfosporosinus- and Clostridia-attributable phylotypes dominated the anoxic enrichment cultures and Alicyclobacillus- and Acidibacillus-attributable phylotypes dominated the oxic enrichment cultures. Our results indicate that while O2 does not impact Fe(III)-reducing activities, it does impact the types of organisms that can be enriched in its presence or absence. The ability of AMD-associated organisms to reduce Fe(III) under both oxic and anoxic conditions may allow them to adapt to fluctuating O2 levels that are encountered in AMD-impacted settings.

The Effect of Dissimilatory Manganese Reduction on Lactate Fermentation and Microbial Community Assembly.

Fermentation and dissimilatory manganese (Mn) reduction are inter-related metabolic processes that microbes can perform in anoxic environments. Fermentation is less energetically favorable and is often not considered to compete for organic carbon with dissimilatory metal reduction. Therefore, the aim of our study was to investigate the outcome of the competition for lactate between fermentation and Mn oxide (birnessite) reduction in a mixed microbial community. A birnessite reducing enrichment culture was obtained from activated sludge with lactate and birnessite as the substrates. This enrichment was further used to test how various birnessite activities (0, 10, 20, and 40 mM) affected the rates of fermentation and metal reduction, as well as community composition. Increased birnessite activity led to a decrease of lactate consumption rate. Acetate and propionate were the main products. With increasing birnessite activity, the propionate/acetate ratio decreased from 1.4 to 0.47. Significant CO2 production was detected only in the absence of birnessite. In its presence, CO2 concentrations remained close to the background since most of the CO2 produced in these experiments was recovered as MnCO3. The Mn reduction efficiency (Mn(II) produced divided by birnessite added) was the highest at 10 mM birnessite added, where about 50% of added birnessite was reduced to Mn(II), whereas at 20 and 40 mM approximately 21 and 16% was reduced. The decreased birnessite reduction efficiency at higher birnessite activities points to inhibition by terminal electron acceptors and/or its toxicity which was also indicated by retarded lactate oxidation and decreased concentrations of microbial metabolites. Birnessite activity strongly affected microbial community structure. Firmicutes and Bacteroidetes were the most abundant phyla at 0 mM of birnessite. Their abundance was inversely correlated with birnessite concentration. The relative sequence abundance of Proteobacteria correlated with birnessite concentrations. Most of the enriched populations were involved in lactate/acetate or amino acid fermentation and the only previously known metal reducing genus detected was related to Shewanella sp. The sequencing data confirmed that lactate consumption coupled to metal reduction was only one of the processes occurring and did not outcompete fermentation processes.

Schwertmannite transformation via direct or indirect electron transfer by a sulfate reducing enrichment culture

Understanding the mechanism of the microbial transformation of Fe(III)-oxyhydroxysulfate minerals is of considerable interest, because this transformation plays an important role in controlling the behaviour of toxic metals from acid mine drainage (AMD). In this study, we examined a sulfate reducing enrichment culture from AMD-contaminated sediments and predicted the possible pathway of electron transfer when incubated with schwertmannite, a common Fe(III)-oxyhydroxysulfate occurring in the AMD environment. Experiments were designed to distinguish the mechanisms by which bacteria facilitate direct (i.e., bacteria allowed to adhere to the mineral) or indirect (i.e., bacteria separated from the mineral by dialysis bag) electron transfer to reduce the mineral. The effects of adding anthraquinone-2,6-disulfonate (AQDS) as an exogenous electron shuttle were also investigated. Vivianite was detected as the main product of schwertmannite transformation. Reduction of sulfate and iron were more pronounced in direct treatments, while more non-reductive dissolution were observed in indirect treatments. The addition of AQDS lead to the production of more dissolved Fe2+ over 20 d than in the absence of AQDS. Microbial community composition differed in direct and indirect treatments, while the addition of AQDS did not significantly affect the community structure in each treatment. After incubation for 20 d, the growth of Desulfovibrio exceeded that of the originally dominant Citrobacter in direct treatments, while an unknown genus most closely related to Citrobacter within Enterobacteriaceae was predominant in indirect treatments. This monodominant community in indirect treatments was assumed not to transfer electron directly to schwertmannite but to rely on shuttling mechanism. PICRUSt results implied that bacteria in indirect treatment have potential to produce shuttling compounds or complexing agents. The absence of dsr genes and the putative fermentative process suggested that the Enterobacteriaceae might indirectly facilitate the dissolution and transformation of schwertmannite.

The biogeochemical fate of nickel during microbial ISA degradation; implications for nuclear waste disposal

Intermediate level radioactive waste (ILW) generally contains a heterogeneous range of organic and inorganic materials, of which some are encapsulated in cement. Of particular concern are cellulosic waste items, which will chemically degrade under the conditions predicted during waste disposal, forming significant quantities of isosaccharinic acid (ISA), a strongly chelating ligand. ISA therefore has the potential to increase the mobility of a wide range of radionuclides via complex formation, including Ni-63 and Ni-59. Although ISA is known to be metabolized by anaerobic microorganisms, the biodegradation of metal-ISA complexes remains unexplored. This study investigates the fate of a Ni-ISA complex in Fe(III)-reducing enrichment cultures at neutral pH, representative of a microbial community in the subsurface. After initial sorption of Ni onto Fe(III)oxyhydroxides, microbial ISA biodegradation resulted in >90% removal of the remaining Ni from solution when present at 0.1 mM, whereas higher concentrations of Ni proved toxic. The microbial consortium associated with ISA degradation was dominated by close relatives to Clostridia and Geobacter species. Nickel was preferentially immobilized with trace amounts of biogenic amorphous iron sulfides. This study highlights the potential for microbial activity to help remove chelating agents and radionuclides from the groundwater in the subsurface geosphere surrounding a geodisposal facility.

Stable Isotope Probing for Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park.

Chocolate Pots hot springs (CP) is a circumneutral-pH Fe-rich geothermal feature located in Yellowstone National Park. Previous Fe(III)-reducing enrichment culture studies with CP sediments identified close relatives of known dissimilatory Fe(III)-reducing bacterial (FeRB) taxa, including Geobacter and Melioribacter However, the abundances and activities of such organisms in the native microbial community are unknown. Here, we used stable isotope probing experiments combined with 16S rRNA gene amplicon and shotgun metagenomic sequencing to gain an understanding of the in situ Fe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled and 13C-labeled acetate to target active FeRB. We searched reconstructed genomes for homologs of genes involved in known extracellular electron transfer (EET) systems to identify the taxa involved in Fe redox transformations. Known FeRB taxa containing putative EET systems (Geobacter, Ignavibacteria) increased in abundance under acetate-amended conditions, whereas genomes related to Ignavibacterium and Thermodesulfovibrio that contained putative EET systems were recovered from incubations without electron donor. Our results suggest that FeRB play an active role in Fe redox cycling within Fe-Si oxide-rich deposits located at the hot spring vent.IMPORTANCE The identification of past near-surface hydrothermal environments on Mars emphasizes the importance of using modern Earth environments, such as CP, to gain insight into potential Fe-based microbial life on other rocky worlds, as well as ancient Fe-rich Earth ecosystems. By combining stable carbon isotope probing techniques and DNA sequencing technology, we gained insight into the pathways of microbial Fe redox cycling at CP. The results suggest that microbial Fe(III) oxide reduction is prominent in situ, with important implications for the generation of geochemical and stable Fe isotopic signatures of microbial Fe redox metabolism within Fe-rich circumneutral-pH thermal spring environments on Earth and Mars.

Fe(III) Reducing Microorganisms from Iron Ore Caves Demonstrate Fermentative Fe(III) Reduction and Promote Cave Formation

ABSTRACTThe banded iron formations (BIF) of Brazil are composed of silica and Fe(III) oxide lamina, and are largely covered by a rock cap of BIF fragments in a goethite matrix (canga). Despite both BIF and canga being highly resistant to erosion and poorly soluble, >3,000 iron ore caves (IOCs) have formed at their interface. Fe(III) reducing microorganisms (FeRM) can reduce the Fe(III) oxides present in the BIF and canga, which could account for the observed speleogenesis. Here, we show that IOCs contain a variety of microbial taxa with member species capable of dissimilatory Fe(III) reduction, including the Chloroflexi, Acidobacteria and the Alpha- Beta- and Gammaproteobacteria; however, Fe(III) reducing enrichment cultures from IOCs indicate the predominance of Firmicutes and Enterobacteriaceae, despite varying the carbon/electron donor, Fe(III) type, and pH. We used model-based inference to evaluate multiple candidate hypotheses that accounted for the variation in medium chemistry and culture composition. Model selection indicated that none of the tested variables account for the dominance of the Firmicutes in these cultures. The addition of H2 to the headspace of the enrichment cultures enhanced Fe(III) reduction, while addition of N2 resulted in diminished Fe(III) reduction, indicating that these Enterobacteriaceae and Firmicutes were reducing Fe(III) during fermentative growth. These results suggest that fermentative reduction of Fe(III) may play a larger role in iron-rich environments than expected. Our findings also demonstrate that FeRM are present within the IOCs, and that their reductive dissolution of Fe(III) oxides, combined with mass transport of solubilized Fe(II) by groundwater, could contribute to IOC formation.

Characterization of Fe (III)-reducing enrichment culture and isolation of Fe (III)-reducing bacterium Enterobacter sp. L6 from marine sediment

To enrich the Fe (III)-reducing bacteria, sludge from marine sediment was inoculated into the medium using Fe (OH)3 as the sole electron acceptor. Efficiency of Fe (III) reduction and composition of Fe (III)-reducing enrichment culture were analyzed. The results indicated that the Fe (III)-reducing enrichment culture with the dominant bacteria relating to Clostridium and Enterobacter sp. had high Fe (III) reduction of (2.73±0.13) mmol/L-Fe (II). A new Fe (III)-reducing bacterium was isolated from the Fe (III)-reducing enrichment culture and identified as Enterobacter sp. L6 by 16S rRNA gene sequence analysis. The Fe (III)-reducing ability of strain L6 under different culture conditions was investigated. The results indicated that strain L6 had high Fe (III)-reducing activity using glucose and pyruvate as carbon sources. Strain L6 could reduce Fe (III) at the range of NaCl concentrations tested and had the highest Fe (III) reduction of (4.63±0.27) mmol/L Fe (II) at the NaCl concentration of 4g/L. This strain L6 could reduce Fe (III) with unique properties in adaptability to salt variation, which indicated that it can be used as a model organism to study Fe (III)-reducing activity isolated from marine environment.

Co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron (III)-reducing enrichment culture.

Methanosaeta harundinacea and Methanosarcina barkeri, known as classic acetoclastic methanogens, are capable of directly accepting electrons from Geobacter metallireducens for the reduction of carbon dioxide to methane, having been revealed as direct interspecies electron transfer (DIET) in the laboratory co-cultures. However, whether their co-occurrences are ubiquitous in the iron (III)-reducing environments and the other species of acetoclastic methanogens such as Methanosarcina mazei are capable of DIET are still unknown. Instead of initiating the co-cultures with pure cultures, two-step cultivation was employed to selectively enrich iron (III)-reducing microorganisms in a coastal gold mining river, Jiehe River, with rich iron content in the sediments. First, iron (III) reducers including Geobacteraceae were successfully enriched by 3-months successive culture on amorphous Fe(III) oxides as electron acceptor and acetate as electron donor. High-throughput Illumina sequencing, terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures actively contained the bacteria belong to Geobacteraceae and Bacilli, exclusively dominated by the archaea belong to Methanosarcinaceae. Second, the enrichment cultures including methanogens and Geobacteraceae were transferred with ethanol as alternative electron donor. Remarkably, aggregates were successively formed in the enrichments after three transfers. The results revealed by RNA-based analysis demonstrate that the co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron (III)-reducing enrichment culture. Furthermore, the aggregates, as close physical contact, formed in the enrichment culture, indicate that DIET could be a possible option for interspecies electron transfer in the aggregates.

Quantification of the Microbial Community in Lateritic Deposits

Lateritic deposits contain oxide ores and usually consist of three layers, namely the limonitic, the saprolite and the garnieritic layer. Limonite, which comprises the top lateritic layer, is a homogeneous ore consisting mainly of goethite with which nickel is associated. Microbe-mineral interactions are of interest for biogeochemical cycles. Microorganisms which are able to mediate redox transformation of Fe-oxide containing minerals are of particular interest when effective bioremediation strategies for insoluble hydroxide and oxide minerals are designed. Quantitive real-time polymerase chain reaction (qPCR) is a culture independent method which is used for the quantification of environmental microorganisms. The aim of this work was to enumerate the microorganisms in different lateritic deposits at Moa mine (Holguin, Cuba) by qPCR and also to obtain Fe (III)-reducing enrichment cultures. A higher microbial abundance was detected in the sampling sites 5124 and 5125 and a lower one in the site 5121. Both, Archaea and Bacteria occurred in all samples except for site 5121 where Bacteria were not detected by qPCR. The Geobacteraceae which comprise Fe-(III)-reducing bacteria were detected at all five lateritic ore sites. The results indicate that these bacteria play a role fundamental in the formation of sediments with a number of oxidized or partially oxidized iron compounds such as magnetite, hematite, goethite and various Fe (III)-oxyhydroxides.

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 μM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.

The Synergistic Effects of High Nitrate Concentrations on Sediment Bioreduction

Groundwaters at nuclear sites can be characterized by low pH and high nitrate concentrations (10–100 mM). These conditions are challenging for bioremediation, often inhibiting microbial Fe(III)-reduction which can limit radionuclide migration. Here, sediment microcosms representative of the UK Sellafield site were used to study the influence of variable pH and nitrate concentrations on microbially-mediated TEAP (terminal electron accepting processes) progression. The rate of reduction through the terminal electron accepting cascade NO− 3 > NO− 2 > Mn(IV)/Fe(III) > SO2− 4 at low pH (∼5.5) was slower than that in bicarbonate buffered systems (pH ∼ 7.0), but in the low pH systems, denitrification and associated pH buffering resulted in conditioning of the sediments for subsequent Fe(III) and sulfate-reduction. Under very high nitrate conditions (100 mM), bicarbonate buffering (pH ∼ 7.0) was necessary for TEAP progression beyond denitrification and the reduction of 100 mM nitrate created alkaline conditions (pH 9.5). 16S rRNA gene analysis showed that close relatives of known nitrate reducers Bacillus niacini and Ochrobactrum grignonense dominated the microbial communities in this reduced sediment. In Fe(III)-reducing enrichment cultures from the 100 mM nitrate system, close relatives of the Fe(III)-reducing species Alkaliphilus crotonatoxidans and Serratia liquifaciens were observed. These results highlight that under certain conditions and contrary to expectations, denitrification may support bioreduction via pH conditioning for optimal metal reduction and radionuclide immobilization.

Humic substance-mediated Fe(III) reduction by a fermenting Bacillus strain from the alkaline gut of a humus-feeding scarab beetle larva

Humus-feeding macroinvertebrates play an important role in the transformation of soil organic matter. Their diet contains significant amounts of redox-active components such as iron minerals and humic substances. In soil-feeding termites, acid-soluble Fe(III) and humic acids are almost completely reduced during gut passage. Here, we show that the reduction of Fe(III) and humic acids takes place also in the alkaline guts of scarab beetle larvae. Sterilized gut homogenates of Pachnoda ephippiata no longer converted Fe(III) to Fe(II), indicating an essential role of the gut microbiota in the process. From Fe(III)-reducing enrichment cultures inoculated with highly diluted gut homogenates, we isolated several facultatively anaerobic, alkali-tolerant bacteria that were closely related to metal-reducing isolates in the Bacillus thioparans group. Strain PeC11 showed a remarkable capacity for dissimilatory Fe(III) reduction, both at pH 7 and 10. Rates were strongly stimulated by the addition of the redox mediator 2,6-antraquinone disulfonate and by redox-active components in the fulvic-acid fraction of humus. Although the contribution of strain PeC11 to intestinal Fe(III) reduction in P. ephippiata remains to be further elucidated, our results corroborate the hypothesis that the lack of oxygen and the solubilization of humic substances in the extremely alkaline guts of humivorous soil fauna provide favorable conditions for the efficient reduction of Fe(III) and humic substances by a primarily fermentative microbiota.

Abundance, Distribution, and Activity of Fe(II)-Oxidizing and Fe(III)-Reducing Microorganisms in Hypersaline Sediments of Lake Kasin, Southern Russia

The extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducing Bacteria and Archaea in pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter(-1)) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries for Bacteria and Archaea revealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 10(2) to 8.32 × 10(3) iron(II)-oxidizing Bacteria and 4.16 × 10(2) to 2.13 × 10(3) iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of total Bacteria, total Archaea, and species dominating the iron(III)-reducing enrichment cultures (relatives of Halobaculum gomorrense, Desulfosporosinus lacus, and members of the Bacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.

The effect of sub-optimal temperature on specific sulfidogenic activity of mesophilic SRB in an H 2-fed membrane bioreactor

The sulfidogenic activity of two mesophilic sulfate reducing enrichment cultures was studied in H 2-fed membrane bioreactors. The two enrichment cultures had different origins; one of them was a mesophilic and the other a psychrotolerant mesophilic culture. The operational temperatures of the reactors were gradually changed: for one the temperature was increased from 9 to 30 °C and for the other it was decreased from 35 to 9 °C. The specific sulfidogenic activities were 21–31, 52–53 and 57–92 mmol SO 4 2− g VSS −1 d −1 at 9, 15 and 30–35 °C, respectively. The sulfate reduction rate of the SRB stabilized to a lower level after the temperature was decreased. The percent electron flow to sulfate reduction was on average 24–32, 50 and 47–69% at 9, 15 and 30–35 °C, respectively. The capability of mesophilic SRB to oxidize electron donor decreased as the temperature was decreased. The results indicate that starting of the reactor operation at 9 °C resulted in higher sulfidogenic activity at sub-optimal temperatures and selective enrichment of the psychrotolerant species improved. The start-up of the reactor at 35 °C resulted in decreased sulfidogenic activity as the temperature was decreased. This indicates that the operational temperature of bioreactors with mesophilic SRB can be decreased to 15–20 °C and the sulfidogenic activity will decrease by 10–40%. Moreover, an operational temperature of 9 °C seems to be close to the lower limit of active sulfate reduction for the mesophilic enrichment cultures used in this study.

Effect of microbial mediated iron plaque reduction on arsenic mobility in paddy soil

The potential of microbial mediated iron plaque reduction, and associated arsenic (As) mobility were examined by iron reducing bacteria enriched from As contaminated paddy soil. To our knowledge, this is the first time to report the impact of microbial iron plaque reduction on As mobility. Iron reduction occurred during the inoculation of iron reducing enrichment culture in the treatments with iron plaque and ferrihydrite as the electron acceptors, respectively. The Fe(II) concentration with the treatment of anthraquinone-2, 6-disulfonic acid (AQDS) and iron reducing bacteria increased much faster than the control. Arsenic released from iron plaque with the iron reduction, and a significant correlation between Fe(II) and total As in culture was observed. However, compared with control, the increasing rate of As was inhibited by iron reducing bacteria especially in the presence of AQDS. In addition, the concentrations of As(III) and As(V) in abiotic treatments were higher than those in the biotic treatments at day 30. These results indicated that both microbial and chemical reductions of iron plaque caused As release from iron plaque to aqueous phase, however, microbial iron reduction induced the formation of more crystalline iron minerals, leading to As sequestration. In addition, the presence of AQDS in solution can accelerate the iron reduction, the As release from iron plaque and subsequently the As retention in the crystalline iron mineral. Thus, our results suggested that it is possible to remediate As contaminated soils by utilizing iron reducing bacteria and AQDS.

PH Gradient-Induced Heterogeneity of Fe(III)-Reducing Microorganisms in Coal Mining-Associated Lake Sediments

Lakes formed because of coal mining are characterized by low pH and high concentrations of Fe(II) and sulfate. The anoxic sediment is often separated into an upper acidic zone (pH 3; zone I) with large amounts of reactive iron and a deeper slightly acidic zone (pH 5.5; zone III) with smaller amounts of iron. In this study, the impact of pH on the Fe(III)-reducing activities in both of these sediment zones was investigated, and molecular analyses that elucidated the sediment microbial diversity were performed. Fe(II) was formed in zone I and III sediment microcosms at rates that were approximately 710 and 895 nmol cm(-3) day(-1), respectively. A shift to pH 5.3 conditions increased Fe(II) formation in zone I by a factor of 2. A shift to pH 3 conditions inhibited Fe(II) formation in zone III. Clone libraries revealed that the majority of the clones from both zones (approximately 44%) belonged to the Acidobacteria phylum. Since Acidobacterium capsulatum reduced Fe oxides at pHs ranging from 2 to 5, Acidobacteria might be involved in the cycling of iron [corrected]. PCR products specific for species related to Acidiphilium revealed that there were higher numbers of phylotypes related to cultured Acidiphilium or Acidisphaera species in zone III than in zone I. From the PCR products obtained for bioleaching-associated bacteria, only one phylotype with a level of similarity to Acidithiobacillus ferrooxidans of 99% was obtained. Using primer sets specific for Geobacteraceae, PCR products were obtained in higher DNA dilutions from zone III than from zone I. Phylogenetic analysis of clone libraries obtained from Fe(III)-reducing enrichment cultures grown at pH 5.5 revealed that the majority of clones were closely related to members of the Betaproteobacteria, primarily species of Thiomonas. Our results demonstrated that the upper acidic sediment was inhabited by acidophiles or moderate acidophiles which can also reduce Fe(III) under slightly acidic conditions. The majority of Fe(III) reducers inhabiting the slightly acidic sediment had only minor capacities to be active under acidic conditions.

Characterization of Fe(III)-reducing enrichment cultures and isolation of Fe(III)-reducing bacteria from the Savannah River site, South Carolina

The Savannah River site, South Carolina (SRS), has been subjected to heavy metal and radionuclide contamination. Dissimilatory Fe(III)-reducing bacteria, by reducing insoluble Fe(III) to soluble Fe(II), may enhance contaminant mobility through subsurface environments. In order to investigate populations of the indigenous iron-reducing microbes from the SRS, duplicate enrichment cultures were initiated using a 10% inoculum of 7 sediment/soil samples, and serial dilutions were made into Wolfe's minimal salts media amended with 50 mM Fe(III) floc, 10 mM acetate and 0.01% yeast extract. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to generate fingerprints of the cultures and track changes in the microbial communities through the dilutions. Cluster analysis determined the relatedness of individual fingerprints. Initial enrichment cultures exhibited complex fingerprints consisting of many individual T-RF peaks, and demonstrated low similarity between sites. After four serial dilutions the fingerprints were less complex and clustered at higher similarities. Several individual T-RF peaks became dominant in a majority of the fingerprints. Cloning and sequence analysis revealed the presence of microbes closely related to Clostridium and Bacillus species and to known iron reducers such as Geobacter species and Pantoea agglomerans. Several Fe(III)-reducing isolates related to Aeromonas, Bacillus and Clostridium species were obtained.

Isolation, characterization, and U(VI)-reducing potential of a facultatively anaerobic, acid-resistant Bacterium from Low-pH, nitrate- and U(VI)-contaminated subsurface sediment and description of Salmonella subterranea sp. nov.

A facultatively anaerobic, acid-resistant bacterium, designated strain FRCl, was isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment at site FW-024 at the Natural and Accelerated Bioremediation Research Field Research Center in Oak Ridge, Tenn. Strain FRCl was enriched at pH 4.5 in minimal medium with nitrate as the electron acceptor, hydrogen as the electron donor, and acetate as the carbon source. Clones with 16S ribosomal DNA (rDNA) sequences identical to the sequence of strain FRCl were also detected in a U(VI)-reducing enrichment culture derived from the same sediment. Cells of strain FRCl were gram-negative motile regular rods 2.0 to 3.4 micro m long and 0.7 to 0.9 microm in diameter. Strain FRCl was positive for indole production, by the methyl red test, and for ornithine decarboxylase; it was negative by the Voges-Proskauer test (for acetylmethylcarbinol production), for urea hydrolysis, for arginine dihydrolase, for lysine decarboxylase, for phenylalanine deaminase, for H(2)S production, and for gelatin hydrolysis. Strain FRCl was capable of using O(2), NO(3)(-), S(2)O(3)(2-), fumarate, and malate as terminal electron acceptors and of reducing U(VI) in the cell suspension. Analysis of the 16S rDNA sequence of the isolate indicated that this strain was 96.4% similar to Salmonella bongori and 96.3% similar to Enterobacter cloacae. Physiological and phylogenetic analyses suggested that strain FRCl belongs to the genus Salmonella and represents a new species, Salmonella subterranea sp. nov.